The mechanisms by which an olfactory sensory neuron expresses a single allele of the approximately 1000 odorant receptor (OR) genes is not known. To design experiments that address directly the mechanism of OR gene choice it is essential to first identify the elements involved in OR gene expression. In a recent study it was shown, for two mouse OR genes, that small tagged genomic fragments ectopically integrated in the mouse genome (transgenes) reproduce the properties of OR gene expression. These fragments contain a homeodomain and an O/E-like site that are conserved in putative promoter regions of many OR genes in mouse, rat, human, and zebrafish. The first aim of this application is to determine if the conserved homeodomain and O/E-like sites have a role in OR gene expression. Essential residues of conserved homeodomain and O/E-like sequences present in the putative promoter region of the M71 OR gene have been mutated. The effect of these mutations has been studied in isolation from the endogenous M71 locus (in transgenic mice), and in the context of the endogenous M71 locus (in gene-targeted knock-in mice). The transgenic and knock-in approaches combined have uncovered important roles for the conserved homeodomain and O/E-like sites in OR gene expression. Here, the role of these sites will be further defined by generating new point mutations that do not abolish the binding properties of these sites but reduce or alter their binding specificity. While carrying out these studies the discrepancies between the transgenic and knock-in results have suggested that additional sequences contribute to the expression of M71. Concurrently, additional homeodomain and O/E-like sites have been identified about 800 bp upstream of the conserved homeodomain and O/E-like sites. The second aim of this application is to test the functional role of these sites in expression of M71. For this purpose the combined transgenic and knock-in approaches described will be used. The in vivo studies proposed here should continue to provide novel insights into the regulation of OR gene expression.